Peritoneal dialysis (PD) is an effective and beneficial treatment for patients with end-stage renal disease;[1],[2] however, peritoneal fibrosis remains a serious complication of long-term PD patients, and a major cause of PD failure.[3],[4],[5] Peritoneal fibrosis usually occurs in response to a variety of insults (including bioincompatible glucose dialysate fluids, peritonitis, uremic toxins, and chronic inflammation).[6] Epithelial–mesenchymal transition (EMT) has been identified as a key mechanism of peritoneal fibrosis in vitro and in PD patients.[5],[7],[8] Various studies have suggested that drugs and peptides could be useful for inhibiting EMT; however, these studies are limited as they block only one signaling pathway. Since multiple signaling pathways are involved in the process of EMT, blocking one signaling pathway may not be enough to prevent EMT. Unfortunately, there are no inhibitors currently available that can simultaneously block two or more signaling pathways that contribute to EMT.

In the past decades, alteration of protein expression was a regularly used strategy for regulating the functions of a protein. More recently, emerging studies have suggested that posttranslational glycosylation modification of proteins plays a key role in altering protein functions, exerting profound effects on many important physiological and pathological processes, including cell growth, migration, and differentiation.[9],[10],[11],[12] Core fucosylation (CF), which is catalyzed by α-1,6 fucosyltransferase (Fut8) in mammals,[13] is an important posttranslational glycosylation found to play a crucial role in pathological processes, including emphysema,[14],[15],[16] schizophrenia,[17],[18] and hepatocellular carcinoma.[19] We recently demonstrated that diminishing the CF of transforming growth factor-β (TGF-β) receptors (TGF-βRs) blocked renal tubular EMT in cultured human renal proximal tubular epithelial cells in vitro and alleviated renal interstitial fibrosis in rats with unilateral ureteral obstruction.[20],[21] However, it is currently unknown how CF may affect EMT in peritoneal fibrosis.

TGF-β is the strongest profibrotic factor in EMT of peritoneal mesothelial cells (PMCs) and a key mediator in dialysis-related peritoneal fibrosis.[5],[22] Platelet-derived growth factor (PDGF) also induced partial EMT in vivo and stimulated human PMC proliferation in vitro.[23],[24]As TGF-βR and PDGF receptors (PDGFR) are both glycosylated, and CF is their common posttranslational modification,[25] we believed that blocking CF may simultaneously inhibit TGF-β and PDGF signaling pathways, resulting in a synergistic protective effect in ameliorating EMT.

In this study, we first hypothesized that CF participated in EMT. With this in mind, we investigated CF expression in rat PMCs cultured in high-glucose (HG) solution. Next, we hypothesized that blocking CF would prevent rat PMC EMT. For this purpose, we established a knockdown model of rat PMCs in vitro to observe the effect. Finally, we examined the activation of TGF-β/Smad2/3 signaling and PDGF/extracellular signal-regulated kinase (ERK) signaling after blocking CF. Our results suggest that CF plays a crucial role during EMT and that blocking it attenuates EMT by simultaneously suppressing the activation of TGF-β and PDGF signaling pathways.

Methods

Cell culture and treatments

Rat PMCs were isolated and cultured according to a previously described method.[5] Briefly, rat PMCs were obtained by infusing 30 ml 0.25% trypsinase–0.2% EDTA–Na2 into the rat abdominal cavity; after 2 h, the fluid was collected under sterile conditions. Next, the cellular components were isolated by centrifugation at 1400 rpm for 5 min, washed with Dulbecco's modified Eagle's medium (DMEM)/F12 medium, and suspended in culture medium supplemented with 15% (v/v) fetal bovine serum (FBS). The rat PMCs used in the experiments were derived from the second to fourth passages, and were incubated with serum-free medium for 24 h to arrest and synchronize the cell growth before each experiment. Next, the medium was changed to fresh 2% FBS–DMEM/F12 medium containing normal glucose (5.6 mmol/L) or HG (2.5% solution, 126 mmol/L) for 3 and 7 days with an exchange of medium every 2–3 days.

Lens culinaris agglutinin–fluorescein isothiocyanate (LCA-FITC; Vector Laboratories, Burlingame, USA) was used to detect the expression of core fucose; and staining was performed as described previously.[10],[20]

Design, preparation, and transfection of the α-1,6 fucosyltransferase small interfering RNA

The siRNAs targeting rat Fut8 and a scrambled siRNA were synthesized by GenePharma (Shanghai, China). The siRNA sequences were validated using BLAST and the rat genome database to evaluate possible cross-reactivity, as described in our previous study.[10],[20] Four siRNAs were synthesized and pooled, and dried siRNA pools were reconstituted in diethyl pyrocarbonate-treated water to a final concentration of 30 nmol/L and stored at −20°C until further use. For transfection, cells were incubated for 24 h to allow multiplication. Then, the siRNAs and transfection reagent were complexed as recommended by the manufacturer, and added to the cell culture.

Real-time reverse transcription polymerase chain reaction

Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed according to our protocol as detailed previously.[20] During analysis of the results, gene expression levels of the target sequence were normalized in relation to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase to control variations in the amount of DNA available for PCR in different samples. All samples were analyzed in triplicate.

Lectin blotting of the immunoprecipitates of ALK-5 (TGF-βRI), TGF-βRII, PDGFRα, and PDGFRβ was performed according to our protocol, as detailed previously.[20]

Western blotting

After rat PMCs were harvested and lysed in RIPA buffer, the lysates were centrifuged at 12,000 ×g for 20 min at 4°C, and then the supernatant was collected. A bicinchoninic acid protein assay kit from Pierce (Madison, WI, USA) was used to determine the protein concentrations. Protein samples were denatured at 100°C for 5 min, separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes (Bio-Rad). Next, the blots were probed with the appropriate primary antibodies at 4°C overnight to detect the expression of ALK-5, TGF-βRII, Smad2/3, phosphorylated (p)-Smad2/3, Fut8, PDGFRα and PDGFRβ, ERK, and phosphorylated (p)-ERK. The blots were then incubated with horseradish peroxidase-labeled secondary antibody for 1 h at 25°C, followed by detection with electrochemiluminescence. Band intensity was quantified for analysis using LabWorks™ Image Analysis software (UVP, Upland, USA).

Statistical analysis

All data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using Student's t-test with SPSS version 13.0 software (SPSS, Chicago, USA). A value of P < 0.05 was considered statistically significant.

The inhibition of EMT of mesothelial cells is an effective method to ameliorate peritoneal fibrosis; however, past studies focused on blocking one signaling pathway to inhibit EMT. CF, catalyzed by Fut8, is a common posttranslational modification in mammalian N-glycans[13] that regulates protein functions and has profound effects on physiological or pathological processes. TGF-βRs and PDGFRs both involve glycosylation and are fucosylated. Inhibiting CF may interfere with the function of aforementioned receptors and suppress the activities of the two signaling pathways. However, it is not clear whether core fucose exists in PMCs, so we first examined it by immunofluorescence staining and found that it existed in rat PMCs. We next investigated the role of CF during rat PMC EMT. The results showed that CF expression was upregulated during rat PMC EMT induced by HG stimulation, and inhibiting it with Fut8 siRNA attenuated EMT, suggesting that CF plays a role in EMT.

Furthermore, we explored the underlying mechanism. Since CF can regulate the function of cell signaling receptors, we then examined the activities of key cell signaling pathways. TGF-β has long been considered a key mediator of EMT and peritoneal fibrosis; TGF-β signals through receptor complexes consisting of TGF-βRI and TGF-βRII, and the activated receptors phosphorylate and activate the Smad proteins. Previously, we confirmed that CF modified TGF-βRs, and that diminishing their CF had protective effects in renal tubular cell EMT in vitro.[20] In the present study, we designed and synthesized four Fut8 siRNAs and transfected them into rat PMCs to knock down Fut8 expression. Using this approach, we blocked the TGF-βR CF [Figure 4], and successfully restored α-SMA and E-cadherin expression, which also prevented the typical fibrotic morphological changes [Figure 3]. Suppressing TGF-β/Smad2/3 signaling activation might be an effective and specific route for therapeutic intervention against EMT; therefore, we examined Smad2/3 phosphorylation, the markers of TGF-β/Smad2/3 signaling activation. We found that inhibiting TGF-βR CF blocked Smad2/3 phosphorylation in vitro, causing the loss of TGF-β/Smad2/3 signaling activation [Figure 4]. The results indicate that inhibiting CF blocks EMT by suppressing TGF-β signaling.

As specifically blocking TGF-βR CF is very difficult, we cannot exclude the possibility that other target proteins of Fut8 fucosylation (such as PDGFR) might be dysfunctional after Fut8 knockdown. That means that the actions of Fut8 siRNA on these signaling molecules could have contributed to its protective effect in EMT. Therefore, we examined PDGFR and found that the PDGFRs were also upregulated, with increased CF in rat PMCs induced by HG stimulation [Figure 5]. Fut8 siRNA effectively inhibited their CF levels [Figure 5], with successful restoration of EMT. The results suggest that PDGFRs play a key role in EMT, and that blocking their CF is beneficial. We next examined ERK phosphorylation, the PDGFR/ERK signaling activation marker, and found that inhibiting PDGFR CF also blocked ERK phosphorylation [Figure 5]. Our results indicate that inhibiting CF blocked EMT by simultaneously suppressing TGF-β and PDGF signaling pathways. As CF is a common posttranslational modification of many glycoproteins, we will explore other glycoproteins involved in peritoneal fibrosis in the future studies to clarify whether they are also affected by the inhibition of CF. In addition, the expression of α-SMA is less in 7 days after cultured in HG solution than in 3 days. We think the reason may be that rat PMCs were in apoptosis process, resulting in smaller cells and more significant spindle shape and less expression of α-SMA. Therefore, this is another reason that we treated rat PMCs with HG solution for 3 days in the remaining in vitro experiments.

In summary, the present study shows that inhibiting CF successfully suppresses the activation of TGF-β/Smad and PDGF/ERK signaling pathways and attenuates rat PMC EMT in vitro. Our results suggest that regulating CF is a potential therapeutic target in peritoneal fibrosis.

Financial support and sponsorship

This work was supported by a grant from the National Natural Science Foundation of China (No. 81530021).